Additional file 9: of Segmental duplications and evolutionary acquisition of UV damage response in the SPATA31 gene family of primates and humans

Tests of SPATA31 antibody specificity. A) Test for antibody specificity on bacterially expressed SPATA31 protein. The C-terminal peptide of SPATA31 (starting at aa position 1122, 3362 bp from the M/ATG start codon) was cloned in reverse and forward orientation into the PGEX-4 T1 bacterial expression vector (see scheme) and transformed into Bl21 E.coli cells for expression analysis. Coomassie blue staining (above) and Western blot analysis (below) of IPTG induced (1 mM and 0.1 mM for 3 h) cells are shown. A strong antibody signal occurred only for the forward orientation under strong induction. B) Western blot of comparative analysis of SPATA31 protein, stained with affinity purified polyclonal SPATA31 antibody (1:500), from human (HFF cells), macaque cells (CV1) and mouse cells (L929). Cells were fractionated by Qproteom (Qiagen) subcellular fractionation kit, loaded on a 4–15% gradient PAGE-SDS gel (Biorad) and electro-blotted onto a PVDF membrane. Note that the picture shows the cytoplasmic fraction, since this yielded a stronger signal. Mouse monoclonal α-tubulin antibody (Sigma 1:1000) was used as loading control. The predicted gene lengths of the protein coding regions of SPATA31 genes were 157kD for the A type (A1) and 130kD for the C type (C1) respectively. The detected size on the western blots shows protein bands around 170–200 kD, probably due to post-translational modifications. C) CV1 cells were transiently transfected with C-terminally Flag-tagged SPATA31A1 (exon4 only, lacking a nuclear localization signal). 24 h after transfection, cells were fixed with cold (4 °C) 1.5% paraformaldehyde at room temperature for 10 min, followed by fixation with Methanol−20 °C for 10 min in−20 °C and stained with DAPI (blue), a monoclonal antibody against Flag-tag (anti-flag-M2 (Sigma, 1:500) (green) and the polyclonal antibody against SPATA31 protein (red). The polyclonal antibody stains both, the protein from cytoplasm expressed Exon4 construct, as well as the endogenous nuclear SPATA31 protein. The scheme of the constructed mammalian expression vector map (pFlag5.1 (Sigma)) is presented below. The figures are merged and magnification is 63X. (PDF 2649 kb)